All-trans retinoic acid (ATRA) is a potential therapeutic agent for the treatment of hematopoietic malignancies, because of its function as an inducer of terminal differentiation of leukemic blasts. Although the efficacy of ATRA as an anticancer drug has been demonstrated by the successful treatment of acute promyelocytic leukemia (APL), the molecular mechanisms of ATRA-induced cell cycle arrest of myeloid cells have not been fully investigated. In this study, we show that the onset of ATRA-induced G(0)/G(1) arrest of human monoblastic U-937 cells is linked to a sharp down-regulation of c-Myc and cyclin E levels and an increase in p21(WAF1/CIP1) expression. This is followed by an increase in p27(Kip1) protein expression due to enhanced protein stability. The importance of an early decrease in Myc expression for these events was demonstrated by the failure of a U-937 subline with constitutive exogenous expression of v-Myc to cell cycle arrest and regulate cyclin E and p27(Kip1) in response to ATRA. Preceding the initiation of G(1) arrest, a transient rise in retinoblastoma protein (pRb), p107, and cyclin A levels was detected. Later, a rapid fall in the levels of cyclins A and B and a coordinate dephosphorylation of pRb at Ser780, Ser795, and Ser807/811 coincided with the accumulation of cells in G(1). These results thus identify a decrease in c-Myc and cyclin E levels and a posttranscriptional up-regulation of p27(Kip1) as important early changes, and position them in the complex chain of events regulating ATRA-induced cell cycle arrest of myeloid cells.